Calcium (Ca) ions act as a regulatory signal for a myriad of cellular processes, and disruption of calcium homeostasis within cells results in numerous pathological conditions. Ca is a messenger molecule in cell signaling that is released in response to numerous stimuli in nearly all cell types. Maintenance of intracellular Ca homeostasis is essential for effective cellular function and perturbation of Ca handling can result in cell death and tissue degeneration. Indeed, disruption of Ca homeostasis has been implicated in the pathogenesis of a variety of diseases including cancer, heart disease, diabetes and neurodegenerative disorders.
Two principal sources of Ca participate in regulating intracellular Ca homeostasis; a) the store of Ca located within sequestering organelles, primarily the endoplasmic reticulum (ER), and b) the vast reservoir of extracellular Ca. Ca stored within the ER is released in a controlled fashion through Ca channels, which are usually proteins from the Inositol 1,4,5-trisphosphate (IP3) receptor or ryanodine receptor (RyR) families. The Ca is then returned into the ER through the action of Ca pumps. The intracellular stores of Ca are replenished from the extracellular reservoir through store-operated Ca entry (SOCE). SOCE is a fairly ubiquitous phenomenon. It has been observed in operation in skeletal muscle (Pan, Z. et al. 2002. Nat. Cell. Biol. 4:379-383), in fibroblasts (Miyakawa, T. et al. 1998. Biochem. J. 329:107-114), and in epithelial cells (Vanden Abeele, F. et al. 2003. J. Biol. Chem. 278:15381-15389).
Gingivitis is an inflammatory disease that results primarily from pathogenic bacteria found within the mouth. It is a condition that arises due to poor oral hygiene but also as a result of certain disease states, in particular those associated with inflammatory states. Although treatment with antibiotics and oral intervention are usual treatments, even aggressive treatment of the bacterial infection fails to reduce gingival symptoms in some patients. Several lines of evidence suggest that alteration to Ca homeostasis, and disruption of SOCE in particular, in various cell types found in the mouth effect dental health.
Several reports in the literature reveal that various Ca channel blocker drugs influence dental health. For example, nifedipine and diltiazem, both Ca channel blocking agents used in the treatment of cardiovascular disease, have been linked to an increased risk of gingival enlargement (Miranda, J. et al. 2001. J. Periodontol. 72:605-611; Miranda, J. et al. 2005. J. Clin. Periodontol. 32:294-298). In an attempt to understand the mechanism underlying the association between gingivitis and Ca channel blocking drugs, it was found that the periodontal inflammatory infiltrate in patients treated with nifedipine had elevated numbers of lymphocytes, in particular B lymphocytes, and these cells decreased significantly in number following periodontal treatment (Bullon, P. et al. 2001. J. Clin. Periodontol. 28:897-903). Population-based observational studies have shown that the use of Ca channel blockers in the treatment of heart disease leads to an increased incidence of dental disorders in patients (Ellis, J. S. et al. 1999. J. Periodontal. 70:63-67; Miranda, J. et al. 2001. J. Periodontol. 72:605-611). These data concerning use of drugs that affect Ca homeostasis in cells are direct evidence for a role of Ca ion movement in the pathogenesis of gingivitis.
Specific evidence also suggests that SOCE in particular is involved in the pathogenesis of gingivitis, particularly in response to the presence of bacteria. SOCE has been implicated during infection of the spirochete Treponema denticola, a periodontal pathogen. Protein fractions including outer membrane from T. denticola inhibit inositol phosphate (Yang, P. F. et al. 1998. Infect. Immun. 66:696-702) and intracellular Ca flux in human gingival fibroblasts (Ko, K. et al. 1998. Infect. Immun. 66:703-709). Later research demonstrated that the major surface protein of T. denticola forms complexes on human gingival fibroblast plasma membranes, inhibiting SOCE and reducing ER Ca stores (Wang, Q et al. 2001. J. Biol. Chem. 276:23056-23064). Furthermore, dental disease with an inherited component, such as localized juvenile periodontitis (LJP), is a disease that is associated with an increased risk of gingivitis, as well as being linked to the presence of defective SOCE (Shibata, K. et al. 2000. J. Periodontol. 71:797-802). Therefore, data suggest that SOCE activity modulation is linked to pathogenesis of periodontal disease, specifically gingivitis.
It has now been found that the SOCE pathway is altered in a specific dose- and time-dependent manner in conditions modeling gingivitis. Further, a peptide derived from a mammalian gene has been found capable of restoring operation of SOCE, preventing the effects of gingivitis and other dental diseases.
In addition to an association of dental disease and gingivitis to SOCE activity, homeostasis of intracellular Ca is critical to cell survival. Many apoptotic stimuli are known to alter concentrations of Ca in the cytosol, storage of Ca in the ER, and/or uptake of Ca into the mitochondria (Pan, Z. et al. 2004. J. Biol. Chem. 279:19387-19390). In non-excitable cells, such as the prostate epithelia, elevation of cytosolic Ca usually results from release through Ca stores in the ER or entry from the extracellular Ca reservoir through activation of SOCE. Depletion of ER Ca stores serves as a signal to activate SOCE. Since Ca storage inside the ER is an essential indicator of a cell's proliferative, metabolic and apoptotic status, the retrograde signaling process from ER Ca depletion to SOCE activation is central to a broad range of cellular and physiological functions. As such, coordinated regulatory mechanisms must exist in the cell to ensure tight control of SOCE function. Aberrant function of SOCE could contribute to either the degenerative or tumorigenic nature of human diseases.
Therefore, in addition to applications in the prevention and treatment of dental diseases, any disease process where SOCE is integral to the pathogenic mechanisms could potentially be targeted by agents that modulate activity of SOCE, diseases related to apoptosis, such as cancer. It has now been found that SOCE activity is decreased in aged skeletal muscle and prostate cancer epithelial cells. The peptide of the present invention, which modulates activity of SOCE, would be useful for the treatment of diseases associated with apoptotic processes (e.g., aging, cancer). Moreover, the peptide of the present invention has application to a number of different human pathologies, across a wide variety of different tissues, as long as the pathology involves altered activity of SOCE.